The objective of the proposed research is to determine the chemical reactions that constitute the process of oxidative degradation of hemoglobin in acquired and inherited Heinz body hemolytic anemia. We propose to determine the specific changes of structure in the heme and globin moieties during this process by isolating intermediate and final products and determining their structures. Changes known or suspected to occur in heme are oxidative cleavage of the porphyrin to biliverdin and smaller fragments and formation of covalent adducts with other compounds through the vinyl side chains and methene bridge carbons. Changes known or suspected to occur in globin are oxidation of thiol groups, condensation of a thiol group with a vinyl side chain of heme, and condensation of side chains of aromatic amino acids with free radicals. Since cleavage of the methene bridges in the coupled oxidation of protohemin IX with ascorbic acid results in a heterogenous mixture of products, the symmetrical molecule octaethylhemin will be used to develop methodology and to work out basic reaction pathways. Myoglobin will be used as a model for study of the oxidative cleavage of heme in hemoproteins since only its alpha-methene bridge is cleaved. Methods based on hydrolysis, extraction, chromatography, and electrophoresis will be developed to separate and purify products. The structure of each product will be determined by the methods of organic and physical chemistry, namely elemental analysis and electronic, infrared, nuclear magnetic resonance, and mass spectrometry. Products of oxidative degradation of hemoglobins from different animal species and from normal and abnormal human hemoglobins will be compared to determine the effect of apoprotein on heme cleavage. Compounds known to cause hemolytic anemia in glucose-6-phosphate dehydrogenase-deficiency but do not cause oxidative degradation of purified hemoglobin will be incubated with oxyhemoglobin in the presence of added superoxide ion and hydrogen peroxide. Any denatured hemoglobin resulting from this treatment will be analyzed for abnormal products that contain structures derived from the hemolytic compound. Urinary pigments from Heinz bodies of different origins will be analyzed and identified.